The influenza viruses consist of subtypes designated A, B and C. Influenza viruses possess a segmented, single negative strand RNA genome which encodes 10 polypeptides that are required for the life cycle of the virus. Each of the eight RNA segments of a complete genome is encapsidated with multiple subunits of the nucleocapsid protein (NP) and associated with a few molecules of the trimeric polymerase (PB1, PB2 and PA subunits), thereby forming the ribonucleoprotein complex (RNP) (Bibliography entry 1). Surrounding these structures is a layer of the matrix protein (M1), which appears to serve as a nexus between the core and the viral envelope. This host cell-derived envelope is studded with the two major virally encoded surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), and a much smaller amount of a nonglycosylated small protein M2 (1, 2). The HA glycoprotein is cleaved by a protease to form HA1 and HA2.
Influenza viral infection is initiated by the attachment of the surface hemagglutinin to a sialic acid-containing cellular receptor. This first virus-cell interaction induces the uptake of the viral particle into the cell by receptor-mediated endocytosis. Under the low pH conditions of the endosome, the HA undergoes a conformational change that facilitates the interaction of the hydrophobic NH2 terminal domain of HA2 and the endosomal membrane, resulting in membrane fusion and subsequent release of the core RNPs and matrix protein (M1) into the cytosol. Disassociation of the RNPs and matrix proteins occurs in the cytosol before the RNPs are translocated to the nucleus where transcription and replication of the complete genome take place (3, 4).
Following primary transcription, newly synthesized proteins initiate the replication of the viral genome which in turn increases transcription and protein synthesis. At this point of the virus life cycle, the surface glycoproteins HA and NA start to accumulate at discrete areas of the plasma membrane from where newly assembled virus will be released. Virus assembly is assumed to begin via some sort of interaction between the cytoplasmic and/or transmembrane domains of the membrane anchored proteins and the underlying matrix protein (M1), which in turn maintains a close association with the RNPs (5, 6). Collectively, HA, NA, M1 and M2 constitute the four virally encoded structural proteins. The contacts between matrix protein M1 and the RNP complexes, as well as the mechanism by which a complete set of eight RNPs gets incorporated into the mature virion particle, have not been well defined. Specific molecular contacts among the structural components are assumed to dictate how the process of morphogenesis initiates and progresses to the point of mature particle assembly and budding from the surface of the host cell.
The complexity of the process has given rise to issues such as: 1) The identification of which viral proteins are required for assembly and budding. 2) The type of protein-protein and lipid-protein interactions between the surface and underlying components which drive the assembly and budding process. 3) The mechanisms by which the RNPs are brought into the assembly site, incorporated into the particle and sorted out from analogous segments. 4) The nature and stoichiometry of the interactions and the regulation of assembly and budding. All of these events occur in a complex cellular environment where some host molecules may or may not enhance or interfere with the progression of the assembly and budding process. This, in turn, leads to the issues of whether cellular proteins are indeed involved in viral assembly and how non-viral proteins are generally excluded from the surface of the budded particles.
A large number of studies have been conducted to address some of these issues with enveloped RNA viruses of different families (5); however most of these issues remain unresolved with respect to influenza virus. Studies with non-segmented RNA virus families (such as the Rhabdoviridae and Paramyxoviridae), which are somewhat morphologically and evolutionarily related to influenza, have shown that the matrix protein (M) of the Rhabdovirus Vesicular stomatitis virus (VSV) by itself is capable of pinching off (budding) from the cell surface as membrane particles (7, 8). In addition, the importance of M proteins in budding is also reflected in the fact that copies of rabies viruses (another Rhabdovirus) with a deletion of the gene encoding the G surface protein are still formed and released from the infected cells (9). More recent work with PIV-3 (a Paramyxovirus) has also shown that the matrix proteins together with nucleocapsid protein (NP) are able to associate into a virus-like structure and bud from the cell surface (10). With respect to influenza virus however, expression of these two proteins using Semliki Forest virus replicons did not show either association between these proteins or budding of membrane vesicles (11).
Performing reverse genetics of influenza virus has been a useful approach to investigate protein-protein interactions between structural components. The importance of the cytoplasmic domain of the glycoproteins in the assembly of influenza has been recently studied (12, 13, 14), and it has been shown that deletion of the HA tail reduced its incorporation into the particle as well as the efficiency of budding, but did not affect virion morphology. On the other hand, virus with a deleted NA tail showed a filamentous morphology and the incorporation of NA into the envelope was impaired. In addition, double deletions seemed to decrease the efficiency of budding as well as infectivity and changed virion morphology, which was distinguishable from those with tail deletions and from wild-type virus. Although double tail deletions appeared to affect the efficiency of budding and morphology of the virus particles, they did not completely abrogate assembly and exit of virion particles. This suggested that M1 protein is able to direct viral assembly and budding (13).
Similarly, the interactions established between the matrix protein and the plasma membrane seem to be critical for virus assembly and release. However the physical nature of this association, whether the matrix protein is completely embedded into the plasma membrane or merely attached by electrostatic interaction, is an unresolved issue. A recent work addressing this question strongly suggested that matrix and membrane were associated through electrostatic interactions, but it could not be ruled out that a certain amount of M1 may be embedded into the membrane (15). The key role of M1 and M2 proteins in the structure of the mature virion is reflected in the spherical or filamentous morphology of the particles when amino acid substitutions are present in either of these molecules (16).
Virus-like particles (VLPs) have been the subject of much interest in recent years as potential candidates for inclusion in immunogenic compositions. This is because VLPs contain one or more surface proteins displayed in a conformation similar enough to their native conformation so that they can elicit a desired immune response. At the same time, VLPs lack the complement of the genetic material required to produce viral progeny in a host. Therefore, unlike a wild-type virus, VLPs cannot cause an infection with disease symptoms or pathology. For example, two or three proteins of rotavirus (a double stranded RNA virus) have been assembled into VLPs which elicited an immune response (17).
Baculovirus expression systems have been broadly used to investigate morphogenesis and assembly of VLPs of non-enveloped viruses that self-assemble into icosahedral structures (18, 19, 20, 21). Similarly, expression in insect cells of the proteins gag and/or env of different members of the retrovirus family has also been used to study assembly and budding of the core structure of enveloped viruses (22, 23, 24, 25).
There is a need to assess the ability of the baculovirus expression system to produce influenza VLPs. In particular, there is a need to identify the minimal number of influenza virus proteins which will assemble into VLPs and to evaluate the morphology and immunogenicity of those VLPs.